Dissimilar promoters for gene suppression

ABSTRACT

Methods of gene suppression comprise transforming eukaryotic cells with recombinant DNA constructs including promoters with dissimilar expression patterns operably linked to one or more gene suppression elements and, optionally, one or more gene expression elements.

REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-In-Part of U.S. patent applicationSer. No. 11/311,892, “Gene Suppression in Transgenic Plants UsingMultiple Constructs”, filed 19 Dec. 2005, incorporated herein byreference.

FIELD OF THE INVENTION

Disclosed herein are recombinant DNA constructs and methods useful ingene suppression and transgenic plant cells, transgenic plants, andtransgenic seeds containing DNA transferred using such recombinant DNAconstructs and methods.

BACKGROUND

Redenbaugh et al. in “Safety Assessment of Genetically Engineered Fruitsand Vegetables—A Case Study of the Flavr Savr™ Tomato”, CRC Press, Inc.(1992) disclosed introducing an anti-sense DNA construct into a tomatogenome by Agrobacterium transformation to produce gene silencing of thepolygalacturonase (PG) gene. A common characteristic of transferred DNA(T-DNA) in transgenic plants exhibiting the desired trait was two ormore T-DNA regions or fragments inserted in a head to head and/or tailto tail arrangement consistent with a report by Jorgensen et al. Mol.Gen. Genet. 207:471-477 (1987) that multiple copies of the T-DNA areoften transferred to and integrated into the genome of a single cell;and, when this occurs, the T-DNAs are predominately organized ininverted repeat structures in plants transformed with Agrobacterium.With reference to FIG. 1 and Table 1 tomato was transformed with aplasmid containing the anti-sense construct (FIG. 1 a) comprising afull-length PG cDNA in the anti-sense orientation between an “enhanced”35S CaMV promoter and the 3′ region of the Agrobacterium tml gene and anartificial kan marker gene. TABLE 1 Element Reference Left border fromT-DNA of pTiA6, Barker et al., Plant Mol. Biol. 2: 335-350 (1983) Mas 5′from mannopine synthase gene, Barker et al., ibid promoter Npt IIneomycin phosphotransferase gene from transposon Tn5, Jorgenson, Mol.Gen. 177: 65 (1979) Mas 3′ polyadenylation region from mannopinesynthase gene Barker et al., ibid Double Gardner et al., Nucl. AcidsRes. 9: 2871-2888 (1981) CaMV35S promoter Anti-sense full length ofpolygalacturonase cDNA in anti-sense PG orientation, Sheehy et al. Proc.Natl. Acad. Sci. USA. 85: 8805-8809 Tml 3′ polyadenylation region of tmlgene from pTiA6, Barker et al. ibid Right border with overdrive t-strandenhancer element, McBride et al. Plant, Mol. Biol. 14: 269-276 (1990)

This construct was used for commercial-scale transformations of severalinbred tomato lines as part of the development and marketing of FlavrSavr™ tomatoes by Calgene in 1994. Tomato lines denoted 501, 502, 7B,22B and 28B were transformed with pCGN1436 using disarmed Agrobacteriumtumefaciens. Events were selected based primarily on phenotype, i.e. lowPG enzyme activity in ripe fruit. Approximately 150 transgenic eventplants were produced for each inbred and 573 plants with ripe fruit wereassayed for PG levels. Between 14-25% of those events across all tomatolines had PG activity lowered by 95% or greater and resulted in a totalof 103 events. Of those plants, 84 had enough seed for kanamycingermination assays to determine segregation ratios and 27 events(representing between 3-10 events for each inbred) segregated 3:1 forthe kan gene. Based on preliminary southern analysis, only about 40% ofthe 27 events with 3:1 segregation ratios clearly appeared to have thePGAS gene and kan gene inserted at a single physical locus. Eight ofthose events were chosen for detailed molecular analysis of T-DNA insertstructures based on the availability of homozygous lines. The results ofthose analyses are shown in FIG. 1 b-d, with the finding that all 8events across the inbred lines had T-DNA inserts containing invertedrepeat elements. The data were consistent with event 501-1001 havingonly a single T-DNA insert, but with the tml 3′ region present as aninverted repeat as illustrated in FIG. 1 b. Six events appeared tocontain two T-DNA regions in a “tail to tail” arrangement as illustratedin FIG. 1 c and event 501-1035 had 3 inserts integrated in a mannerillustrated in FIG. 1 d.

Northern analysis of the 8 selected events demonstrated no correlationbetween PG anti-sense RNA levels and the efficacy of PG gene silencing.A range of PG anti-sense RNA levels were observed, ranging from easilydetected amounts in one event to undetectable levels in multiple events,all of which produced the gene silenced trait of delayed ripening.Potential read-through transcripts larger in size than expected weredetected for the marker kan gene and for the PG anti-sense gene. Theobservation that inverted repeat elements in T-DNA inserts were likelytranscribed as larger than expected RNAs, albeit at low levels, supportsthe thesis that PG mRNA reductions were due to RNAi induced by theproduction of RNA capable of forming dsRNA. The structure of anti-senseinsert illustrated in FIG. 1 b with inverted repeat of 3′ tml (sensefollowed by anti-sense) is very similar to the sense construct utilizedfor gene silencing by Brummell et al. as disclosed in Plant Journal, 33,793-800 (2003) using 3′ nos element (anti-sense followed by sense) as aninverted repeat. In each case a 3′ hairpin loop could be formed and usedas primer for RNA-dependent RNA polymerase and the formation of dsRNAsequences of the target RNA.

The discovery of inverted repeats of inserted T-DNA illustrated in FIG.1 c and as an element of FIG. 1 c, suggested increasing the efficacy oftransformation with anti-sense DNA constructs by directly transformingwith the inverted repeat in the plasmid. Yet, the presence of invertedrepeats in plasmids has been believed to be problematic when insidebacteria, e.g. E. coli, which interfere with plasmid maintenance,resulting in plasmid instability. The following described inventionprovides the potential advantages of employing inverted repeat elementsin a transformation construct without the disadvantage of adjacentinverted repeats in bacteria.

A single expression cassette containing inverted repeats of sequencesfrom a target gene may not be effective for gene suppression in desiredplant tissue. For instance, the CaMV 35S promoter is typically denotedas “constitutive”, but is does not express well in pollen. The“constitutive” rice actin 1 promoter expresses well in pollen but not aswell in leaves. The following described invention provides advantages ofgene suppression in multiple plant tissues not afforded by use of asingle cassette with a single promoter.

SUMMARY OF THE INVENTION

This invention provides an improved method of gene suppressioncomprising transforming eukaryotic cells with multiple gene suppressionconstructs located adjacent to each other on a plasmid. In one aspect ofthe invention the multiple gene suppression constructs can be multipleadjacent anti-sense gene suppression constructs; in another aspect theycan be multiple adjacent sense (co-suppression) gene suppressionconstructs. In a further aspect, they can be multiple adjacent sense andanti-sense gene suppression constructs. The multiple adjacent genesuppression constructs can be overlapping or non-overlapping. Moreparticularly the method comprises inserting into a plasmid forAgrobacterium-mediated transformation a cassette for expressing sense(or anti-sense) DNA from a gene targeted for suppression adjacent to asecond cassette for expressing the same sense (or anti-sense) DNA.

The invention further provides transgenic seed having in its genome arecombinant DNA construct comprising: (a) a plant endosperm-specificpromoter operably linked to at least one first gene suppression element,and (b) a plant embryo-specific promoter in the opposite orientation tothe plant endosperm-specific promoter and located 3′ to the at least onefirst gene suppression element.

The invention further provides stably transgenic plant cells having intheir genome a recombinant DNA construct comprising: (a) a firstpromoter operably linked to at least one first gene suppression elementfor silencing at least one first target gene, and (b) a second promoterthat is in the opposite orientation to the first promoter and is located3′ to the at least one first gene suppression element, wherein the firstand the second promoters have dissimilar expression patterns, andwherein transcription of the recombinant DNA construct in a plant cellresults in silencing of the at least one first target gene.

This invention further provides constructs for transformation ofeukaryotic cells (such as plant cells), methods for their use, andstably transformed transgenic plant cells containing such constructs.These constructs, include (a) a first promoter operably linked to atleast one first gene suppression element for silencing at least onefirst target gene, and (b) a second promoter that is in the oppositeorientation to the first promoter and is located 3′ to the at least onefirst gene suppression element, wherein the first and said secondpromoters have dissimilar expression patterns, and wherein transcriptionof the recombinant DNA construct in a eukaryotic cell (such as a plantcell) results in silencing of the at least one first target gene. Thedissimilar expression patterns include spatially or temporallydissimilar expression patterns, as well as inducible expressionpatterns.

A characteristic of the invention is variation in regulatory elements inthe cassettes, i.e. the promoter regulatory elements and/or thepolyadenylation regulatory elements. In embodiments using anti-sensecassettes, the first anti-sense expression cassette comprises a firstpromoter operably linked to DNA of a gene targeted for suppression in ananti-sense orientation optionally followed by a first 3′ element (e.g.comprising a polyadenylation signal and polyadenylation site); and, thesecond anti-sense RNA expression cassette comprises a second promoteroperably linked to said DNA of a gene targeted for suppression in ananti-sense orientation optionally followed by a second 3′ element. Thefirst and second cassettes are assembled into a DNA construct in atail-to-tail configuration so that the promoters are at the ends of theassembled construct bounding transcribable DNA of the gene targeted forsuppression and, when 3′ elements are used, the 3′ elements are (a)contiguous or (b) adjacent to the promoters either between the promotersand the transcribable DNA or at the extreme regions of the assembly. Ata minimum the first and second promoters are different. First and second3′ elements can also be different.

The method further comprises transforming eukaryotic cells bytransferring a DNA construct with such assembled first and secondcassettes from a plasmid by Agrobacterium-mediated transformation. Atransgenic organism is regenerated from cells transformed with the firstand second cassettes; and, a trait resulting from suppression of thelevel of protein encoded by said DSA of a gene targeted for suppressionis measured in the transgenic organism.

In aspects of the method promoters can include well-known promoters thatare functional in plants including Agrobacterium nopaline synthase (nos)promoter, Agrobacterium octopine synthase (ocs) promoter, thecauliflower mosaic virus promoter (CaMV 35S), figwort mosaic viruspromoter (FMV), maize RS81 promoter, rice actin promoter, maize RS324promoter, maize PR-1 promoter, maize A3 promoter, gamma coixin B32endosperm-specific promoter, maize L3 oleosin embryo-specific promoter,rd29a promoter, and any of the other well-know promoters useful in plantgene expression.

In aspects of the method the intron is any spliceable intron. In someembodiments, the intron is preferably a transcription-enhancing intron,e.g., “enhancers” such as 5′ introns of the rice actin 1 and rice actin2 genes, the maize alcohol dehydrogenase gene, the maize heat shockprotein 70 gene, and the maize shrunken 1 gene.

In aspects of the method the 3′ elements are selected from the groupconsisting of the well-known 3′ elements, e.g. Agrobacterium gene 3′elements such as nos 3′, tml 3′, tmr 3′, tms 3′, ocs 3′, tr7 3′ andplant gene 3′ elements such as wheat (Triticum aestivum) heat shockprotein 17 (Hsp17) 3′, a wheat ubiquitin gene 3′, a wheatfructose-1,6-biphosphatase gene 3′, a rice glutelin gene 3′, a ricelactate dehydrogenase gene 3′, a rice beta-tubulin gene 3′, a pea (Pisumsativum) ribulose bisphosphate carboxylase gene (rbs) 3′, and 3′elements from other genes within the host plant.

In other aspects of the method at least one of the multiple cassettescomprises a marker gene, e.g. an herbicide marker gene that providesresistance to glyphosate (aroA or EPSPS) or glufosinate (pat or bar); abacteriocide marker gene that provides resistance to kanamycin (npt II),gentamycin (aac 3), hygromycin (aph IV), streptomycin and spectinomycin(aadA), or ampicillin (amp); or a screenable marker such as a luciferase(luc) or a fluorescent protein (gfp) or a beta-glucuronidase (uidA). Thelength of the DNA of a gene targeted for suppression can be any length,but preferably at least 21 nucleotides in length.

Another aspect of the invention provides a plasmid forAgrobacterium-mediated transformation comprising such a first cassettefor expressing sense (or anti-sense) DNA from a gene targeted forsuppression adjacent to such a second cassette for expressing the sameDNA, where the cassettes are assembled so that the different 3′untranslated regions are contiguous. In many cases the cassettes and atleast one marker cassette are located between left and right T-DNAborders on the plasmid.

In a preferred aspect of the invention a transgenic corn plant containsa DNA construct with adjacent cassettes for anti-sense suppression ofthe lysine ketoglutarate reductase gene using an endosperm specificpromoter in one cassette and an embryo specific promoter in the othercassette.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 illustrate DNA constructs.

FIG. 3 depicts non-limiting examples of constructs of the invention,e.g., as described in Example 3. The endosperm-specific promoter isindicated by “pB32”, the embryo-specific promoter by “pL3”, the genesuppression element(s) by “SUP-LKR/SDH” (which represents a stabilizedanti-sense suppression element targetting endogenous lysineketoglutarate reductase/saccharopine dehydrogenase), “GSE1”, and “GSE2”,and terminators by “tHsp17”, “tGlb1”, “ter1”, and “ter2”.

DETAILED DESCRIPTION OF THE INVENTION

As used herein “cassette” means a combination of DNA elements normallyassociated with the expression of protein from a gene and comprises atleast (a) DNA for initiating transcription such as a promoter element,(b) DNA coding for a protein such as cDNA or genomic DNA comprisingexons and introns, and (c) DNA for splicing 3′ RNA from transcribed RNAafter coding sequence and adding a polyA tail such as a 3′ elementcontaining a polyadenylation site. Typically, when the DNA coding for aprotein is in a sense orientation, the transcribed RNA can be translatedto express protein or, in some cases, for sense co-suppression. When theDNA coding for protein is in an anti-sense orientation, the transcribedRNA can be involved in a gene suppression mechanism. For instance, topromote gene suppression anti-sense DNA typically corresponds to DNAthat is transcribed to mRNA upstream of a polyadenylation site. Thus, an“anti-sense cassette” means a combination of DNA elements comprising apromoter operably linked to anti-sense oriented DNA from a gene targetedfor suppression and a 3′ element. Although common, it is not criticalthat the 3′ element contain a polyadenylation site. What is important ineither adjacent sense cassettes or adjacent anti-sense cassettes is thatadjacent 3′ elements are distinct, i.e. transcribed RNA from adjacent 3′elements is are not capable of hybridizing to from double-stranded RNAor being readily excised from a plasmid in E. coli.

Recombinant DNA constructs, e.g. the cassettes of this invention, can bereadily prepared by those skilled in the art using commerciallyavailable materials and well-known, published methods. When multiplegenes are targeted for suppression, polycistronic DNA elements can befabricated as illustrated and disclosed in U.S. application Ser. No.10/465,800, incorporated herein by reference. A useful technology forbuilding DNA constructs and vectors for transformation is the GATEWAY™cloning technology (available from Invitrogen Life Technologies,Carlsbad, Calif.) uses the site specific recombinase LR cloning reactionof the Integrase att system from bacteriophage lambda vectorconstruction, instead of restriction endonucleases and ligases. The LRcloning reaction is disclosed in U.S. Pat. Nos. 5,888,732 and 6,277,608,U.S. Patent Application Publications 2001283529, 2001282319,20020007051, and 20040115642, all of which are incorporated herein byreference. The GATEWAY™ Cloning Technology Instruction Manual which isalso supplied by Invitrogen also provides concise directions for routinecloning of any desired DNA into a vector comprising operable plantexpression elements.

An alternative vector fabrication method employs ligation-independentcloning as disclosed by Aslandis, C. et al., Nucleic Acids Res., 18,6069-6074, 1990 and Rashtchian, A. et al., Biochem., 206, 91-97, 1992where a DNA fragment with single-stranded 5′ and 3′ ends are ligatedinto a desired vector which can then be amplified in vivo.

Numerous promoters that are active in plant cells have been described inthe literature. These include promoters present in plant genomes as wellas promoters from other sources, including nopaline synthase (NOS)promoter and octopine synthase (OCS) promoters carried on tumor-inducingplasmids of Agrobacterium tumefaciens, caulimovirus promoters such asthe cauliflower mosaic virus or figwort mosaic virus promoters. Forinstance, see U.S. Pat. Nos. 5,858,742 and 5,322,938 which discloseversions of the constitutive promoter derived from cauliflower mosaicvirus (CaMV35S), U.S. Pat. No. 5,378,619 which discloses a FigwortMosaic Virus (FMV) 35S promoter, U.S. Pat. No. 6,437,217 which disclosesa maize RS81 promoter, U.S. Pat. No. 5,641,876 which discloses a riceactin promoter, U.S. Pat. No. 6,426,446 which discloses a maize RS324promoter, U.S. Pat. No. 6,429,362 which discloses a maize PR-1 promoter,U.S. Pat. No. 6,232,526 which discloses a maize A3 promoter, U.S. Pat.No. 6,177,611 which discloses constitutive maize promoters, U.S. Pat.No. 6,433,252 which discloses a maize L3 oleosin promoter, U.S. Pat. No.6,429,357 which discloses a rice actin 2 promoter and intron, U.S. Pat.No. 5,837,848 which discloses a root specific promoter, U.S. Pat. No.6,084,089 which discloses cold inducible promoters, U.S. Pat. No.6,294,714 which discloses light inducible promoters, U.S. Pat. No.6,140,078 which discloses salt inducible promoters, U.S. Pat. No.6,252,138 which discloses pathogen inducible promoters, U.S. Pat. No.6,175,060 which discloses phosphorus deficiency inducible promoters,U.S. Patent Application Publication 2002/0192813A1 which discloses 5′,3′ and intron elements useful in the design of effective plantexpression vectors, U.S. patent application Ser. No. 09/078,972 whichdiscloses a coixin promoter, U.S. patent application Ser. No. 09/757,089which discloses a maize chloroplast aldolase promoter, and U.S. patentapplication Ser. No. 10/739,565 which discloses water-deficit induciblepromoters, all of which are incorporated herein by reference. These andnumerous other promoters that function in plant cells are known to thoseskilled in the art and available for use in recombinant polynucleotidesof the present invention to provide for expression of desired genes intransgenic plant cells.

In aspects of the method the 3′ elements are selected from the groupconsisting of the well-known 3′ elements from Agrobacterium tumefaciensgenes such as nos 3′, tml 3′, tmr 3′, tms 3′, ocs 3′, tr7 3′, e.g.disclosed in U.S. Pat. No. 6,090,627, incorporated herein by reference;3′ elements from plant genes such as wheat (Triticum aestivum) heatshock protein 17 (Hsp17 3′), a wheat ubiquitin gene, a wheatfructose-1,6-biphosphatase gene, a rice glutelin gene a rice lactatedehydrogenase gene and a rice beta-tubulin gene, all of which aredisclosed in U.S. published patent application 2002/0192813 A1,incorporated herein by reference; and the pea (Pisum sativum) ribulosebisphosphate carboxylase gene (rbs 3′), and 3′ elements from the geneswithin the host plant.

Furthermore, the promoters may be altered to contain multiple “enhancersequences” to assist in elevating gene expression. Such enhancers areknown in the art. By including an enhancer sequence with suchconstructs, the expression of the selected protein may be enhanced.These enhancers often are found 5′ to the start of transcription in apromoter that functions in eukaryotic cells, but can often be insertedin the forward or reverse orientation 5′ or 3′ to the coding sequence.In some instances, these 5′ enhancing elements are introns. Particularlyuseful enhancers are the 5′ introns of the rice actin 1 (see U.S. Pat.No. 5,641,876) and rice actin 2 genes, the maize alcohol dehydrogenasegene, the maize heat shock protein 70 gene (see U.S. Pat. No. 5,593,874)and the maize shrunken 1 gene.

In some aspects of the invention it is preferred that the promoterelement in the DNA construct be capable of causing sufficient expressionto result in the production of an effective amount of a polypeptide inwater deficit conditions. Such promoters can be identified and isolatedfrom the regulatory region of plant genes that are over expressed inwater deficit conditions. Specific water-deficit-inducible promoters foruse in this invention are derived from the 5′ regulatory region of genesidentified as a heat shock protein 17.5 gene (HSP17.5), an HVA22 gene(HVA22), a Rab17 gene and a cinnamic acid 4-hydroxylase (CA4H) gene(CA4H) of Zea mays, or derived from the 5′ regulatory region of genesidentified as a rab17 gene (RAB17), a cinnamic acid 4-hydroxylase (CA4H)gene (CA4H), an HVA22 gene (HVA22), and genes for heat shock proteins17.5 (HSP17.5), 22 (HSP22) and 16.9 (HSP16.9) of Oryza sativa. Suchwater-deficit-inducible promoters are disclosed in U.S. application Ser.No. 10/739,565 and Ser. No. 11/066,911, incorporated herein byreference.

In other aspects of the invention, sufficient expression in plant seedtissues is desired to effect improvements in seed composition. Exemplarypromoters for use for seed composition modification include promotersfrom seed genes such as napin (U.S. Pat. No. 5,420,034), maize L3oleosin (U.S. Pat. No. 6,433,252), zein Z27 (Russell et al. (1997)Transgenic Res. 6(2): 157-166), globulin 1 (Belanger et al (1991)Genetics 129:863-872), glutelin 1 (Russell (1997) supra), andperoxiredoxin antioxidant (Per1) (Stacy et al. (1996) Plant Mol Biol.31(6):1205-1216).

In still other aspects of the invention, preferential expression inplant green tissues is desired. Promoters of interest for such usesinclude those from genes such as SSU (Fischhoff et al. (1992) Plant MolBiol. 20:81-93), aldolase and pyruvate orthophosphate dikinase (PPDK)(Taniguchi et al. (2000) Plant Cell Physiol. 41(1):42-48).

In practice DNA is introduced into only a small percentage of targetcells in any one transformation experiment. Marker genes are used toprovide an efficient system for identification of those cells that arestably transformed by receiving and integrating a transgenic DNAconstruct into their genomes. Preferred marker genes provide selectivemarkers which confer resistance to a selective agent, such as anantibiotic or herbicide. Any of the herbicides to which plants of thisinvention may be resistant are useful agents for selective markers.Potentially transformed cells are exposed to the selective agent. In thepopulation of surviving cells will be those cells where, generally, theresistance-conferring gene is integrated and expressed at sufficientlevels to permit cell survival. Cells may be tested further to confirmstable integration of the exogenous DNA. Commonly used selective markergenes include those conferring resistance to antibiotics such askanamycin (nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4)or resistance to herbicides such as glufosinate (bar or pat) andglyphosate (EPSPS). Examples of such selectable markers are illustratedin U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047, all ofwhich are incorporated herein by reference. Screenable markers whichprovide an ability to visually identify transformants can also beemployed, e.g., a gene expressing a colored or fluorescent protein suchas a luciferase or green fluorescent protein (GFP) or a gene expressinga beta-glucuronidase or uidA gene (GUS) for which various chromogenicsubstrates are known.

The invention provides transgenic seed having in its genome arecombinant DNA construct comprising: (a) a plant endosperm-specificpromoter operably linked to at least one first gene suppression element,and (b) a plant embryo-specific promoter in the opposite orientation tothe plant endosperm-specific promoter and located 3′ to the at least onefirst gene suppression element. In some embodiments, the plantembryo-specific promoter can transcribe the at least one first genesuppression element. In other embodiments, the plant embryo-specificpromoter can transcribe at least one second gene suppression element(e.g., a second gene suppression element for silencing the same genetargetted by the endosperm-specific promoter, or for silencing adifferent gene).

In one embodiment of the transgenic seed, the at least one first genesuppression element includes a gene suppression element for silencing acatabolism gene of an amino acid (or of an amino acid's biosyntheticintermediates), such as, but not limited to, a lysine catabolism gene.Other catabolism genes can be silenced, such as genes involved incatabolism of lipids or carbohydrates or of their biosyntheticintermediates. In one specifically claimed embodiment, the transgenicseed is transgenic maize seed, and the amino acid catabolism gene is alysine catabolism gene, such as the endogenous maize LKR/SDH gene.

In some embodiments of the transgenic seed, the recombinant DNAconstruct further includes one or more elements selected from: (a) atleast one second gene suppression element operably linked to the plantembryo-specific promoter; (b) an amino acid biosynthesis gene operablylinked to either the plant endosperm-specific promoter or plantembryo-specific promoter; and (c) a selectable marker gene. In someembodiments where both a gene suppression element and an expressionelement for another gene (e.g., an amino acid biosynthesis gene) areoperably linked to one promoter, the gene suppression element can beembedded in an intron, which in many embodiments is preferably atranscription-enhancing intron (e.g., “enhancers” such as 5′ introns ofthe rice actin 1 and rice actin 2 genes, the maize alcohol dehydrogenasegene, the maize heat shock protein 70 gene, and the maize shrunken 1gene). In some preferred embodiments, the recombinant DNA constructfurther comprises one or more elements selected from: (a) at least onesecond gene suppression element for silencing a lysine catabolism geneoperably linked to the plant embryo-specific promoter; (b) a lysinebiosynthesis (e.g., an exogenous DHDPS or CordapA gene) biosynthesisgene operably linked to the plant endosperm-specific promoter; (c) anaspartate kinase gene (e.g., a lysC gene) operably linked to either theplant endosperm-specific promoter or plant embryo-specific promoter; and(d) a selectable marker gene. In certain preferred embodiments, theconstruct includes an aspartate kinase gene (operably linked to eitherthe embryo- or the endosperm-specific promoter) and a gene suppressionelement for silencing endogenous LKR/SDH (preferably operably linked tothe endosperm-specific promoter or to both the embryo- and theendosperm-specific promoters), and preferably also includes an exogenousDHDPS or CordapA gene (operably linked to the endosperm-specificpromoter). Marker genes include selectable markers (such as are commonlyused to select transformed cells, e.g., antibiotic or herbicideresistance genes), detectable markers (e.g., luciferase, greenfluorescent protein, GUS), and can include coding sequence or non-codingsequence (for example a suppression element that suppresses anendogenous gene resulting in an observable phenotype, e.g., asuppression element for silencing a gene involved in plant pigmentproduction).

FIG. 3 depicts non-limiting embodiments of recombinant DNA constructsuseful for providing transgenic seeds of the invention:

(a) (see FIG. 3A) the recombinant DNA construct includes: (i) a plantendosperm-specific promoter operably linked to at least one first genesuppression element including DNA that transcribes to RNA for silencinga lysine catabolism gene by forming double-stranded RNA (e.g., DNA thatincludes at least one anti-sense DNA segment that is anti-sense to atleast one segment of the at least one target gene and at least one senseDNA segment that is at least one segment of the at least one firsttarget gene; or DNA that encodes at least one trans-acting miRNA andthat transcribes to double-stranded RNA targetted to the target gene inboth transcriptional directions), and (ii) a plant embryo-specificpromoter in the opposite orientation to the first promoter and operablylinked to the at least one first gene suppression element;

(b) (see FIG. 3B) the recombinant DNA construct includes: (i) a plantendosperm-specific promoter operably linked to at least one first genesuppression element including DNA that transcribes to RNA for silencinga lysine catabolism gene by forming double-stranded RNA (e.g., DNA thatincludes at least one anti-sense DNA segment that is anti-sense to atleast one segment of the at least one target gene and at least one senseDNA segment that is at least one segment of the at least one firsttarget gene; or DNA that encodes a trans-acting miRNA in bothtranscriptional directions), (ii) a plant embryo-specific promoter inthe opposite orientation to the first promoter and operably linked tothe at least one first gene suppression element, and (iii) at least oneterminator operably linked to either the first or second promoters(wherein each terminator can be on either side of the oppositelyoriented promoter); or

(c) (see FIG. 3C) the recombinant DNA construct includes: (i) a plantendosperm-specific promoter operably linked to at least one firstintron-embedded gene suppression element for silencing a lysinecatabolism gene, at least one lysine biosynthesis gene (preferablycordapA or lysC or both), and a first terminator, (ii) a plantembryo-specific promoter in the opposite orientation to the firstpromoter and operably linked to at least one second gene suppressionelement (which is optionally embedded in an intron, preferably atranscription-enhancing intron) for silencing a lysine catabolism gene;or

(d) (see FIG. 3D) the recombinant DNA construct includes: (i) a firstgene suppression cassette including a plant endosperm-specific promoteroperably linked to at least one first intron-embedded gene suppressionelement for silencing a lysine catabolism gene, at least one lysinebiosynthesis gene (preferably cordapA or lysC or both), and a firstterminator, and (ii) a second gene suppression cassette including aplant embryo-specific promoter operably linked to at least one secondgene suppression element for silencing a lysine catabolism gene, and asecond terminator, wherein the first and second gene suppressioncassettes are in opposite orientations (optionally assembled so that thepromoters are at the ends of the construct); or

(e) (see FIG. 3E) the recombinant DNA construct includes: (i) a firstgene suppression cassette including a plant endosperm-specific promoteroperably linked to at least one first intron-embedded gene suppressionelement for silencing a lysine catabolism gene, at least one lysinebiosynthesis gene (preferably cordapA or lysC or both), and a firstterminator, and (ii) a second gene suppression cassette including aplant embryo-specific promoter operably linked to at least oneintron-embedded second gene suppression element for silencing a lysinecatabolism gene, at least one lysine biosynthesis gene (preferablycordapA or lysC or both) and a second terminator, wherein the first andsecond gene suppression cassettes are in opposite orientations(optionally assembled so that the promoters are at the ends of theconstruct); or

(f) (see FIG. 3F) the recombinant DNA construct includes: (i) a firstgene suppression cassette including a plant endosperm-specific promoteroperably linked to at least one first gene suppression element forsilencing a lysine catabolism gene, and a first terminator, and (ii) asecond gene suppression cassette including a plant embryo-specificpromoter operably linked to at least one second gene suppression elementfor silencing a lysine catabolism gene, and a second terminator, whereinthe first and second gene suppression cassettes are in oppositeorientations (optionally assembled so that the promoters are at the endsof the construct).

FIG. 4 depicts other specific embodiments of constructs of theinvention. While FIGS. 3 and 4 depict some gene suppression elements asincluding sense and anti-sense sequence in the form of a stabilizedanti-sense element (“SUP-LKR/SDH”), other gene suppression elements areuseful, providing that they are transcribed by the appropriate promoterto an RNA molecule or molecules capable of suppressing the targetgene(s). Where constructs include two non-overlapping expression“cassettes” (see, for example, FIGS. 3D, 3E, and 3F), an alternativearrangement is for the two promoters to be located adjacent to eachother and oppositely oriented (resulting in “divergent” transcription).

Generally, it is preferable to prevent “read through” of terminators andunintentional silencing of, e.g., an opposing promoter or sequenceoperably linked to an opposing promoter. Thus, in some embodiments, anintron or other spliceable element such as a ribozyme can be optionallyinserted (see, for example, the bottom construct of FIG. 3B) to prevent“read through” of any downstream sequence. In some embodiments where atranscript of a gene suppression element need not be polyadenylated(e.g., where the target is localized in the nucleus), an intron or otherspliceable element such as a ribozyme can be inserted to prevent “readthrough” of the opposing promoter (see, for example, the bottomconstruct of FIG. 3A). In other embodiments, an intron can be arrangedto include a gene suppression element embedded within it, and further toprevent “read through” of any downstream sequence (see, for example, thebottom construct of FIG. 3E).

The invention further provides stably transgenic plant cells having intheir genome a recombinant DNA construct including: (a) a first promoteroperably linked to at least one first gene suppression element forsilencing at least one first target gene, and (b) a second promoter thatis in the opposite orientation to the first promoter and is located 3′to the at least one first gene suppression element, wherein the firstand the second promoters have dissimilar expression patterns, andwherein transcription of the recombinant DNA construct in a plant cellresults in silencing of the at least one first target gene. By “stablytransgenic plant cells” is meant plant cells that have stably integratedan exogenous gene (transgene) into their genome. In many preferredembodiments, such stably transgenic plant cells are homozygous for thetransgene. In particularly preferred embodiments, the integratedtransgene is heritable, that is, transferable to progeny plants. Thedissimilar expression patterns include spatially or temporallydissimilar expression patterns, as well as inducible expressionpatterns. Non-limiting examples of suitable first and second promotersinclude first and second promoters that control transcription indifferent organelles, cells, or tissues, or first and second promotersthat control transcription under different times (e.g., at differentpoints of a circadian cycle) or developmental periods, or first andsecond promoters that are induced differently by an inducer or areinduced by different inducers. The stably transgenic plant cells can beisolated transgenic plant cells or can be in a transgenic plantregenerated from the transgenic plant cell, or a transgenic progeny seedor transgenic progeny plant of such a regenerated transgenic plant. Inone preferred embodiment of the stably transgenic plant cells, the firstand the second promoters comprise a plant embryo-specific promoter and aplant endosperm-specific promoter and the stably transgenic plant cellscomprise seed embryo and endosperm cells of a crop plant (e.g., maize,rice, or other crop plants that have seed containing substantialendosperm).

This invention further provides constructs for transformation ofeukaryotic cells (such as plant cells and animal cells), methods fortheir use, and stably transgenic plant cells containing such constructs.These constructs include (a) a first promoter operably linked to atleast one first gene suppression element for silencing at least onefirst target gene, and (b) a second promoter that is in the oppositeorientation to the first promoter and is located 3′ to the at least onefirst gene suppression element, wherein the first and said secondpromoters have dissimilar expression patterns, and wherein transcriptionof the recombinant DNA construct in a eukaryotic cell (such as a plantcell or animal cell) results in silencing of the at least one firsttarget gene. The dissimilar expression patterns include spatially ortemporally dissimilar expression patterns, as well as inducibleexpression patterns. Thus, in some embodiments, the first and secondpromoters have dissimilar spatial expression patterns, and the silencingoccurs in at least two distinct spatial locations. In other embodiments,the first and second promoters have dissimilar temporal expressionpatterns, and the silencing occurs in at least two distinct times ordevelopmental stages (either non-overlapping or overlapping periods oftime). Suitable promoters include, for example, first and secondpromoters that control transcription in different organelles (e.g.,plastids, nucleus, mitochondria), cells, or tissues, or first and secondpromoters that control transcription under different times (e.g., atdifferent points of a circadian cycle) or developmental periods, orfirst and second promoters that are induced differently by an inducer orare induced by different inducers.

In some embodiments of the recombinant DNA construct, the at least onegene suppression element is under transcriptional control of both thefirst and the second promoters. In these embodiments, the at least onegene suppression element is transcribed in both directions andsuppresses the at least one target gene in two locations (or at twodistinct times or developmental stages).

In some embodiments, the recombinant DNA construct further includes oneor more of: (a) a second gene suppression element operably linked to thesecond promoter; (b) at least one gene expression element for expressingat least one exogenous gene; (c) at least one terminator, and (d) atleast one T-DNA border. The second gene suppression element is arrangedsuch that transcription of the second gene suppression element resultsin the intended silencing of the gene it targets; thus, in manyembodiments, the second gene suppression element is oriented opposite tothe first promoter. The at least one exogenous gene expressed by the atleast on gene expression element can be any gene or genes to beexpressed out of native context, and can include, e.g., a marker gene, acodon-optimized gene, an allelic replacement of a native gene.

In some embodiments of the recombinant DNA construct, the at least onefirst gene suppression element includes at least one element selectedfrom the group consisting of: (a) DNA that includes at least oneanti-sense DNA segment that is anti-sense to at least one segment of theat least one first target gene; (b) DNA that includes multiple copies ofat least one anti-sense DNA segment that is anti-sense to at least onesegment of the at least one first target gene; (c) DNA that includes atleast one sense DNA segment that is at least one segment of the at leastone first target gene; (d) DNA that includes multiple copies of at leastone sense DNA segment that is at least one segment of the at least onefirst target gene; (e) DNA that transcribes to RNA for suppressing theat least one first target gene by forming double-stranded RNA andincludes at least one anti-sense DNA segment that is anti-sense to atleast one segment of the at least one target gene and at least one senseDNA segment that is at least one segment of the at least one firsttarget gene; (f) DNA that transcribes to RNA for suppressing the atleast one first target gene by forming a single double-stranded RNA andincludes multiple serial anti-sense DNA segments that are anti-sense toat least one segment of the at least one first target gene and multipleserial sense DNA segments that are at least one segment of the at leastone first target gene; (g) DNA that transcribes to RNA for suppressingthe at least one first target gene by forming multiple double strands ofRNA and includes multiple anti-sense DNA segments that are anti-sense toat least one segment of the at least one first target gene and multiplesense DNA segments that are at least one segment of the at least onefirst target gene, and wherein the multiple anti-sense DNA segments andthe multiple sense DNA segments are arranged in a series of invertedrepeats; (h) DNA that includes nucleotides derived from a plant miRNA;(i) DNA that includes nucleotides of a siRNA; (j) DNA that transcribesto an RNA aptamer capable of binding to a ligand; and (k) DNA thattranscribes to an RNA aptamer capable of binding to a ligand, and DNAthat transcribes to regulatory RNA capable of regulating expression ofthe first target gene, wherein the regulation is dependent on theconformation of the regulatory RNA, and the conformation of theregulatory RNA is allosterically affected by the binding state of theRNA aptamer. Suitable gene suppression elements are further described inU.S. patent application Ser. No. 11/303,745, which is incorporatedherein by reference.

In some embodiments of the recombinant DNA construct, the first genesuppression element is embedded in an intron. In preferred embodiments,the intron is flanked on one or on both sides by non-protein-coding DNA,and more preferably is a transcription-enhancing intron (e.g.,“enhancers” such as 5′ introns of the rice actin 1 and rice actin 2genes, the maize alcohol dehydrogenase gene, the maize heat shockprotein 70 gene, and the maize shrunken 1 gene).

In some embodiments, the recombinant DNA construct further includes asecond gene suppression element operably linked to the second promoter,wherein the first and second gene suppression elements are embedded inan intron (either individually in separate introns or together in asingle intron). The second gene suppression element is arranged suchthat transcription of the second gene suppression element results in theintended silencing of the gene it targets; thus, in many embodiments,the second gene suppression element is oriented opposite to the firstpromoter.

In one particularly preferred embodiment of the recombinant DNAconstruct, the first and the second promoters include a plantembryo-specific promoter and a plant endosperm-specific promoter.

Further provided by this invention is a method of gene silencing in aplant, including: (a) transforming a plant cell with the recombinant DNAconstruct including (i) a first promoter operably linked to at least onefirst gene suppression element for silencing at least one first targetgene, and (ii) a second promoter that is in the opposite orientation tothe first promoter and is located 3′ to the at least one first genesuppression element, wherein the first and said second promoters havedissimilar expression patterns, and wherein transcription of therecombinant DNA construct in a eukaryotic cell (such as a plant cell oranimal cell) results in silencing of the at least one first target gene,thereby providing a transgenic plant cell; (b) preparing a regeneratedtransgenic plant from the transgenic plant cell, or a transgenic progenyseed or transgenic progeny plant of the regenerated transgenic plant;(c) transcribing the recombinant DNA construct in the regeneratedtransgenic plant or the transgenic progeny seed or transgenic progenyplant, whereby the at least one first target gene is silenced in theregenerated transgenic plant or the transgenic progeny seed ortransgenic progeny plant.

In preferred embodiments of the method, the plant is a crop plant, forexample, grain crops (e.g., maize, rice, wheat, barley, rye), legumes(e.g., soybean, alfalfa, beans, peanuts), oilseeds (e.g., rape, canola,soybean, nuts), and fruit or vegetable crop plants. In one preferredembodiment, the recombinant DNA construct is transcribed in a transgenicprogeny seed having substantial endosperm (e.g., a transgenic maize orrice seed or other cereal grain seed), and the first and the secondpromoters include a plant embryo-specific promoter and a plantendosperm-specific promoter. Particularly preferred is the methodwherein the transgenic progeny seed is transgenic progeny maize seed,the at least one first target gene is at least one lysine catabolismgene, and the at least one lysine catabolism gene is silenced in embryoand endosperm cells of the transgenic progeny seed. In anotherparticularly preferred embodiment of the method, the transgenic progenyseed is transgenic progeny maize seed, the at least one first targetgene is at least one lysine catabolism gene, the at least one lysinecatabolism gene is silenced in embryo and endosperm cells of thetransgenic progeny seed, and the recombinant DNA construct furtherincludes at least one lysine biosynthesis gene operably linked to theendosperm-specific promoter.

This invention further provides a method for manufacturing transgenicmaize seed having an increased level of a nutrient, the methodcomprising: (a) selecting a first transgenic maize plant comprising arecombinant DNA construct including (i) a first promoter operably linkedto at least one first gene suppression element for silencing at leastone first target gene, wherein the at least one first target gene is acatabolism gene of a nutrient selected from an amino acid, a lipid, or acarbohydrate, and (ii) a second promoter that is in the oppositeorientation to the first promoter and is located 3′ to the at least onefirst gene suppression element, wherein the first and said secondpromoters have dissimilar expression patterns, and wherein transcriptionof the recombinant DNA construct in a eukaryotic cell (such as a plantcell or animal cell) results in silencing of the at least one firsttarget gene; (b) introgressing the recombinant DNA construct into asecond maize plant; (c) growing seed from the second maize plant toproduce a population of progeny maize plants; (d) screening thepopulation of progeny maize plants for progeny maize plants that producemaize seed having an increased level of the nutrient, relative tonon-transgenic maize plants; (e) selecting from the population one ormore progeny maize plants that produce maize seed having an increasedlevel of the nutrient, relative to non-transgenic maize plants; (f)verifying that the recombinant DNA construct is stably integrated in theselected progeny maize plants; (g) verifying that the catabolism gene ofthe nutrient is silenced in the selected progeny maize plants, relativeto maize plants lacking the recombinant DNA construct; (h) collectingtransgenic maize seed from the selected progeny maize plants. Therecombinant DNA construct optionally includes a gene expression element.In various embodiments of the method, the nutrient to be increased is anamino acid (e.g., lysine, methionine, or tryptophan), a lipid (e.g., afatty acid or fatty acid ester), or a carbohydrate (e.g., a simple sugaror a complex carbohydrate). In one preferred embodiment of the method,the nutrient is lysine, the catabolism gene is a lysine catabolism gene(e.g., maize lysine ketoglutarate reductase/saccharopine dehydrogenase),and the first and the second promoters include a plant embryo-specificpromoter and a plant endosperm-specific promoter; optionally, therecombinant DNA construct also includes a gene expression element forexpression of a lysine biosynthesis gene (e.g., cordapA or lysC).

Plant Transformation Methods

Numerous methods for transforming plant cells with recombinant DNA areknown in the art and may be used in the present invention. Two commonlyused methods for plant transformation are Agrobacterium-mediatedtransformation and microprojectile bombardment. Microprojectilebombardment methods are illustrated in U.S. Pat. No. 5,015,580(soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No. 5,538,880(corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No. 6,160,208(corn); U.S. Pat. No. 6,399,861 (corn) and U.S. Pat. No. 6,153,812(wheat) and Agrobacterium-mediated transformation is described in U.S.Pat. No. 5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean); U.S.Pat. No. 5,591,616 (corn); and U.S. Pat. No. 6,384,301 (soybean), all ofwhich are incorporated herein by reference. For Agrobacteriumtumefaciens based plant transformation systems, additional elementspresent on transformation constructs include T-DNA left and/or rightborder sequences (generally both left and right border sequences, butpreferably at least one border sequence, e.g. at least a right bordersequence) to facilitate incorporation of the recombinant polynucleotideinto the plant genome.

In general it is useful to introduce recombinant DNA randomly, i.e. at anon-specific location, in the genome of a target plant line. In specialcases it may be useful to target recombinant DNA insertion in order toachieve site-specific integration, e.g. to replace an existing gene inthe genome, to use an existing promoter in the plant genome, or toinsert a recombinant polynucleotide at a predetermined site known to beactive for gene expression. Several site specific recombination systemsexist which are known to function implants include cre-10× as disclosedin U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S. Pat. No.5,527,695, both incorporated herein by reference.

Transformation methods of this invention are preferably practiced intissue culture on media and in a controlled environment. “Media” refersto the numerous nutrient mixtures that are used to grow cells in vitro,that is, outside of the intact living organism. Recipient cell targetsinclude, but are not limited to, meristem cells, callus, immatureembryos and gametic cells such as microspores, pollen, sperm and eggcells. It is contemplated that any cell from which a fertile plant maybe regenerated is useful as a recipient cell. Callus may be initiatedfrom tissue sources including, but not limited to, immature embryos,seedling apical meristems, microspores and the like. Cells capable ofproliferating as callus are also recipient cells for genetictransformation. Practical transformation methods and materials formaking transgenic plants of this invention, e.g. various media andrecipient target cells, transformation of immature embryos andsubsequent regeneration of fertile transgenic plants are disclosed inU.S. Pat. Nos. 6,194,636 and 6,232,526 and U.S. patent application Ser.No. 09/757,089, which are incorporated herein by reference.

The seeds of transgenic plants can be harvested from fertile transgenicplants and be used to grow progeny generations of transformed plants ofthis invention including hybrid plants line comprising the recombinantDNA construct expressing an agent for genes suppression.

In addition to direct transformation of a plant with a recombinant DNAconstruct, transgenic plants can be prepared by crossing a first planthaving a recombinant DNA construct with a second plant lacking theconstruct. For example, recombinant DNA for gene suppression can beintroduced into a first plant line that is amenable to transformation toproduce a transgenic plant which can be crossed with a second plant lineto introgress the recombinant DNA for gene suppression into the secondplant line.

A transgenic plant with recombinant DNA effecting gene suppression canbe crossed with transgenic plant line having other recombinant DNA thatconfers another trait, e.g. yield improvement, herbicide resistance orpest resistance to produce progeny plants having recombinant DNA thatconfers both gene suppression and the other trait. Typically, in suchbreeding for combining traits the transgenic plant donating theadditional trait is a male line and the transgenic plant carrying thebase traits is the female line. The progeny of this cross will segregatesuch that some of the plants will carry the DNA for both parental traitsand some will carry DNA for one parental trait; such plants can beidentified by markers associated with parental recombinant DNA Progenyplants carrying DNA for both parental traits can be crossed back intothe female parent line multiple times, e.g. usually 6 to 8 generations,to produce a progeny plant with substantially the same genotype as oneoriginal transgenic parental line but for the recombinant DNA of theother transgenic parental line.

EXAMPLES Example 1

This example illustrates a method of this invention. With reference toFIG. 2 two cassettes are prepared for anti-sense suppression ofluciferase in an organism expressing luciferase. A first luciferaseanti-sense cassette comprises CaMV 35S promoter (35S 3′) operably linkedto an anti-sense segment of firefly luciferase coding DNA (anti-senseLUC) and nos 3′ element. A second luciferase anti-sense cassettecomprises a FMV promoter (FMV 5′) operably linked to the same anti-sensesegment of firefly luciferase coding DNA and a wheat heat shock protein3′ element (hsp 3′). The anti-sense cassettes are assembled in antransformation plasmid inverted with respect to each other with therespective 3′ elements being contiguous. Surprisingly, the assembledcassettes are not prone to excision when the plasmid is inserted intocommon strains of E. coli. The plasmid is co-transformed into a plantcell along with a two plasmids capable of expressing the fireflyluciferase and Renilla luciferase genes, the latter serving as abaseline control against which firefly luciferase expression isnormalized. Thus, the ratio of firefly luciferase to Renilla luciferaseexpression is a measurement of the level of suppression of the fireflyluciferase gene. As compared to plant cells transformed with a singlecopy of either of the firefly luciferase anti-sense cassettes, themultiple cassettes exhibit a higher level of firefly luciferasesuppression in transgenic plant cells.

Example 2

This example illustrates a construct useful for selective genesuppression in plant tissues. A first anti-sense gene suppressionconstruct was prepared comprising a corn plant endosperm specificpromoter B32 (nucleotides 848 through 1259 of GenBank accession numberX70153, see also Hartings et al. (1990) Plant Mol. Biol., 14:1031-1040)operably linked to transcribable DNA consisting of about 500 base pairsof the LKR domain of a maize lysine ketoglutarate reductase/saccharopinedehydrogenase gene (LKR/SDH) in first segment in an anti-senseorientation linked to a second segment in a sense orientation. BecauseLKR is a lysine catabolism enzyme, its suppression resulted in increasedlysine. A second anti-sense gene suppression construct was preparedessentially the same as the first anti-sense gene suppression constructexcept that the promoter was replaced with a corn plant embryo specificpromoter L3 oleosin (see U.S. Pat. No. 6,433,252). A third genesuppression construct according to this invention was prepared bylinking a B32 promoter that used in the first construct to the 3′ end ofthe second construct providing a construct with opposing promotersoperably linked to an anti-sense oriented segment of DNA from the genetargeted for suppression. In one alternative embodiment the genesuppression construct of this invention is prepared from the secondanti-sense gene suppression construct by replacing the 3′ regulatoryregion that provides a polyadenylation signal and site with the B32promoter inserted in an opposite orientation to the L3 promoter at theopposing end of the construct (see FIG. 3A). In another alternativeembodiment, the construct of this invention is prepared by adding theB32 promoter downstream of the 3′ regulatory region and in an oppositeorientation to the L3 promoter at the opposing end of the construct;optionally a second 3′ regulatory region is inserted between the L3promoter and the transcribable DNA. In yet another embodiment theconstruct of this invention is prepared by locating 3′ regulatoryregions at the external regions of the construct where each 3′regulatory region is oriented to the promoters at the opposing end ofthe construct. In still another embodiment two anti-sense constructs areassembled in a tail-to-tail orientation providing a construct bounded bythe respective promoters.

Plasmids suitable for Agrobacterium-mediated plant transformation wereprepared using each of (a) the first anti-sense gene suppressionconstruct with the B32 promoter, (b) the second ant-sense genesuppression construct with the L3 promote and (c) a gene suppressionconstruct of this invention with a B32 and an L3 promoter at opposingends of the construct and in opposite orientations. Each construct wasinserted into a plasmid for binary vector of an Agrobacterium-mediatedtransformation system between left and right T-DNA borders and next to aselectable marker cassette for expressing an aroA gene from A.tumefaciens. Each plasmid was inserted into maize callus byAgrobacterium-mediated transformation. Events were selected as beingresistance to glyphosate herbicide and grown into transgenic maizeplants to produce F1 seed. Mature seeds from each event are analyzed todetermine success of transformation and suppression of LKR. The maturetransgenic seeds are dissected to extract protein for Western analysis.Seed from transgenic maize plants shows reduction in LKR and increasedlysine as compared to wild type. The first construct with the endospermspecific promoter provides seed with about 1000 ppm of free lysine; LKRreduction is essentially observed only in endosperm tissue. The secondconstruct with the embryo specific promoter provides seed with about 300ppm of free lysine; LKR reduction is essentially observed only in embryotissue. Because lysine is believed to travel between embryo andendosperm, concurrent suppression of LKR in both embryo and endospermtissues using the construct of this invention provides seed with highervalues of free lysine than the additive effect from suppression in onetissue alone, e.g. greater than 1300 ppm.

Example 3

This non-limiting example illustrates constructs for transforming plantcells and methods for use thereof, and transgenic maize seed of theinvention. In this specific example, a recombinant DNA constructincluding a plant embryo-specific promoter and a plantendosperm-specific promoter, each operably linked to at least one genesuppression element for silencing a lysine catabolism gene, is used toprovide transgenic plant cells, and transgenic progeny maize plants andseeds derived from such transgenic plant cells, wherein the transgenicprogeny seed have increased lysine.

One non-limiting embodiment of a recombinant DNA constructs useful,e.g., for providing transgenic plant cells, transgenic plants, andtransgenic seeds of the invention, is illustrated in FIG. 3B andincludes: (i) a plant endosperm-specific promoter operably linked to atleast one first gene suppression element including DNA that transcribesto RNA for silencing a lysine catabolism gene by forming double-strandedRNA (e.g., DNA that includes at least one anti-sense DNA segment that isanti-sense to at least one segment of the at least one target gene andat least one sense DNA segment that is at least one segment of the atleast one first target gene; or DNA that encodes a trans-acting miRNA inboth transcriptional directions), (ii) a plant embryo-specific promoterin the opposite orientation to the first promoter and operably linked tothe at least one first gene suppression element, and (iii) at least oneterminator operably linked to either the first or second promoters(wherein each terminator can be on either side of the oppositelyoriented promoter.

In a specific example, a recombinant DNA construct (illustrated in FIG.4, third construct from top) is stably introduced byAgrobacterium-mediated transformation into maize plant cell and progenymaize plants are regenerated as described under “Plant TransformationMethods” above. This construct includes (a) a plant endosperm-specificpromoter (“pB32”) operably linked to a stabilized anti-sense genesuppression element targetting endogenous lysine ketoglutaratereductase/saccharopine dehydrogenase (“SUP-LKR/SDH”) embedded in anintron (“intron 1”, e.g., an hsp70 intron) and a gene expression elementfor expressing a lysine-insensitive Corynebacterium DHDPS or cordapA(“cordap A”) (see U.S. Pat. Nos. 6,459,019 and 5,773,691 and U.S. PatentApplication Publication No. 2003/0056242, which are incorporated byreference), and a first terminator; and (b) a plant embryo-specificpromoter (“pL3”) in the opposite orientation to the endosperm-specificpromoter and operably linked to a stabilized anti-sense gene suppressionelement targetting endogenous lysine ketoglutaratereductase/saccharopine dehydrogenase (“SUP-LKR/SDH”), optionally aselectable marker, and a second terminator. Optionally an intron orribozyme is positioned to prevent “read-through” of the oppositepromoter. Transgenic seed from the regenerated plants show reduction inLKR in both embryo and endosperm seed tissues, relative to seed in whichthe recombinant DNA construct is absent or is not transcribed, andincreased levels of lysine in the transgenic seed. Levels of cordapA areincreased, relative to seed in which the recombinant DNA construct isabsent or is not transcribed, resulting in a further increased lysinelevel in the transgenic seed. Overall, levels of lysine in thetransgenic seed are increased relative to transgenic seed in which theexpression of endogenous lysine ketoglutarate reductase/saccharopinedehydrogenase is silenced in either embryo or endosperm tissues but notin both.

1. Transgenic seed having in its genome a recombinant DNA constructcomprising: (a) a plant endosperm-specific promoter operably linked toat least one first gene suppression element, and (b) a plantembryo-specific promoter in the opposite orientation to said plantendosperm-specific promoter and located 3′ to said at least one firstgene suppression element.
 2. The transgenic seed of claim 1, whereinsaid at least one first gene suppression element comprises a genesuppression element for silencing an amino acid catabolism gene.
 3. Thetransgenic seed of claim 2, wherein said recombinant DNA constructfurther comprises one or more elements selected from: (a) at least onesecond gene suppression element operably linked to said plantembryo-specific promoter; (b) an amino acid biosynthesis gene operablylinked to either said plant endosperm-specific promoter or plantembryo-specific promoter; and (c) a selectable marker gene.
 4. Thetransgenic seed of claim 2, wherein said transgenic seed is transgenicmaize seed, and said amino acid catabolism gene is a lysine catabolismgene.
 5. The transgenic seed of claim 4, wherein said recombinant DNAconstruct further comprises one or more elements selected from: (a) atleast one second gene suppression element for silencing a lysinecatabolism gene operably linked to said plant embryo-specific promoter;(b) a lysine biosynthesis gene operably linked to said plantendosperm-specific promoter; (c) an aspartate kinase gene operablylinked to either said plant endosperm-specific promoter or plantembryo-specific promoter; and (d) a selectable marker gene.
 6. Thetransgenic seed of claim 2, wherein: (a) said recombinant DNA constructcomprises: (i) a plant endosperm-specific promoter operably linked to atleast one first gene suppression element comprising DNA that transcribesto RNA for silencing a lysine catabolism gene by forming double-strandedRNA, and (ii) a plant embryo-specific promoter in the oppositeorientation to said first promoter and operably linked to said at leastone first gene suppression element; or (b) said recombinant DNAconstruct comprises: (i) a plant endosperm-specific promoter operablylinked to at least one first gene suppression element comprising DNAthat transcribes to RNA for silencing a lysine catabolism gene byforming double-stranded RNA, (ii) a plant embryo-specific promoter inthe opposite orientation to said first promoter and operably linked tosaid at least one first gene suppression element, and (iii) at least oneterminator operably linked to either said first or second promoters; or(c) said recombinant DNA construct comprises: (i) a plantendosperm-specific promoter operably linked to at least one firstintron-embedded gene suppression element for silencing a lysinecatabolism gene, at least one lysine biosynthesis gene, and a firstterminator, (ii) a plant embryo-specific promoter in the oppositeorientation to said first promoter and operably linked to at least onesecond gene suppression element for silencing a lysine catabolism gene;or (d) said recombinant DNA construct comprises: (i) a first genesuppression cassette comprising a plant endosperm-specific promoteroperably linked to at least one first intron-embedded gene suppressionelement for silencing a lysine catabolism gene, at least one lysinebiosynthesis gene, and a first terminator, and (ii) a second genesuppression cassette comprising a plant embryo-specific promoteroperably linked to at least one second gene suppression element forsilencing a lysine catabolism gene, and a second terminator, whereinsaid first and second gene suppression cassettes are in oppositeorientations; or (e) said recombinant DNA construct comprises: (i) afirst gene suppression cassette comprising a plant endosperm-specificpromoter operably linked to at least one first intron-embedded genesuppression element for silencing a lysine catabolism gene, at least onelysine biosynthesis gene and a first terminator, and (ii) a second genesuppression cassette comprising a plant embryo-specific promoteroperably linked to at least one intron-embedded second gene suppressionelement for silencing a lysine catabolism gene, at least one lysinebiosynthesis gene and a second terminator, wherein said first and secondgene suppression cassettes are in opposite orientations; or (f) saidrecombinant DNA construct comprises: (i) a first gene suppressioncassette comprising a plant endosperm-specific promoter operably linkedto at least one first gene suppression element for silencing a lysinecatabolism gene, and a first terminator, and (ii) a second genesuppression cassette comprising a plant embryo-specific promoteroperably linked to at least one second gene suppression element forsilencing a lysine catabolism gene, and a second terminator, whereinsaid first and second gene suppression cassettes are in oppositeorientations.
 7. Stably transgenic plant cells having in their genome arecombinant DNA construct comprising: (a) a first promoter operablylinked to at least one first gene suppression element for silencing atleast one first target gene, and (b) a second promoter that is in theopposite orientation to said first promoter and is located 3′ to said atleast one first gene suppression element, wherein said first and saidsecond promoters have dissimilar expression patterns, and whereintranscription of said recombinant DNA construct in a plant cell resultsin silencing of said at least one first target gene.
 8. The stablytransgenic plant cells of claim 7, wherein said first and said secondpromoters comprise a plant embryo-specific promoter and a plantendosperm-specific promoter and said stably transgenic plant cellscomprise seed embryo and endosperm cells of a crop plant.
 9. Arecombinant DNA construct for transformation of a plant cell,comprising: (a) a first promoter operably linked to at least one firstgene suppression element for silencing at least one first target gene,and (b) a second promoter that is in the opposite orientation to saidfirst promoter and is located 3′ to said at least one first genesuppression element, wherein said first and said second promoters havedissimilar expression patterns, and wherein transcription of saidrecombinant DNA construct in a plant cell results in silencing of saidat least one first target gene.
 10. The recombinant DNA construct ofclaim 9, wherein first and second promoters have dissimilar spatialexpression patterns, and said silencing occurs in at least two distinctspatial locations.
 11. The recombinant DNA construct of claim 9, whereinfirst and second promoters have dissimilar temporal expression patterns,and said silencing occurs in at least two distinct times ordevelopmental stages.
 12. The recombinant DNA construct of claim 9,wherein said at least one gene suppression element is undertranscriptional control of both said first and said second promoters.13. The recombinant DNA construct of claim 9, further comprising one ormore of: (a) a second gene suppression element operably linked to saidsecond promoter; (b) at least one gene expression element for expressingat least one exogenous gene, (c) at least one terminator, and (d) atleast one T-DNA border.
 14. The recombinant DNA construct of claim 9,wherein said at least one first gene suppression element comprises atleast one element selected from the group consisting of: (a) DNA thatcomprises at least one anti-sense DNA segment that is anti-sense to atleast one segment of said at least one first target gene; (b) DNA thatcomprises multiple copies of at least one anti-sense DNA segment that isanti-sense to at least one segment of said at least one first targetgene; (c) DNA that comprises at least one sense DNA segment that is atleast one segment of said at least one first target gene; (d) DNA thatcomprises multiple copies of at least one sense DNA segment that is atleast one segment of said at least one first target gene; (e) DNA thattranscribes to RNA for suppressing said at least one first target geneby forming double-stranded RNA and comprises at least one anti-sense DNAsegment that is anti-sense to at least one segment of said at least onetarget gene and at least one sense DNA segment that is at least onesegment of said at least one first target gene; (f) DNA that transcribesto RNA for suppressing said at least one first target gene by forming asingle double-stranded RNA and comprises multiple serial anti-sense DNAsegments that are anti-sense to at least one segment of said at leastone first target gene and multiple serial sense DNA segments that are atleast one segment of said at least one first target gene; (g) DNA thattranscribes to RNA for suppressing said at least one first target geneby forming multiple double strands of RNA and comprises multipleanti-sense DNA segments that are anti-sense to at least one segment ofsaid at least one first target gene and multiple sense DNA segments thatare at least one segment of said at least one first target gene, andwherein said multiple anti-sense DNA segments and said multiple senseDNA segments are arranged in a series of inverted repeats; (h) DNA thatcomprises nucleotides derived from a plant miRNA; (i) DNA that comprisesnucleotides of a siRNA; (j) DNA that transcribes to an RNA aptamercapable of binding to a ligand; and (k) DNA that transcribes to an RNAaptamer capable of binding to a ligand, and DNA that transcribes toregulatory RNA capable of regulating expression of said first targetgene, wherein said regulation is dependent on the conformation of saidregulatory RNA, and said conformation of said regulatory RNA isallosterically affected by the binding state of said RNA aptamer. 15.The recombinant DNA construct of claim 9, wherein said first genesuppression element is embedded in an intron.
 16. The recombinant DNAconstruct of claim 9, further comprising a second gene suppressionelement operably linked to said second promoter, wherein said first andsecond gene suppression elements are embedded in an intron.
 17. Therecombinant DNA construct of claim 9, wherein said first and said secondpromoters comprise a plant embryo-specific promoter and a plantendosperm-specific promoter.
 18. A method of gene silencing in a plant,comprising: (a) transforming a plant cell with the recombinant DNAconstruct of claim 9, thereby providing a transgenic plant cell; (b)preparing a regenerated transgenic plant from said transgenic plantcell, or a transgenic progeny seed or plant of said regeneratedtransgenic plant; (c) transcribing said recombinant DNA construct insaid regenerated transgenic plant or said transgenic progeny seed orplant whereby said at least one first target gene is silenced in saidregenerated transgenic plant or said transgenic progeny seed or plant.19. The method of claim 18, wherein said plant is a crop plant.
 20. Themethod of claim 18, wherein said recombinant DNA construct istranscribed in a transgenic progeny seed having substantial endosperm,and said first and said second promoters comprise a plantembryo-specific promoter and a plant endosperm-specific promoter. 21.The method of claim 20, wherein said transgenic progeny seed istransgenic progeny maize seed, said at least one first target gene is atleast one lysine catabolism gene, and said at least one lysinecatabolism gene is silenced in embryo and endosperm cells of saidtransgenic progeny seed.
 22. The method of claim 21, wherein saidrecombinant DNA construct further comprises at least one lysinebiosynthesis gene operably linked to said endosperm-specific promoter.